Name: cell-cell fusion of genome edited cell lines for perturbation of cellular structure and function
Uploaded: 2019-12-07T17:00:00
Description: Watch this Scientific Journal Video about Cell-cell Fusion of Genome Edited Cell Lines for Perturbation of Cellular Structure and Function at JoVE.com

This work was funded by a Wellcome Trust Henry Wellcome Fellowship to R.M. (https://wellcome.ac.uk/grant number 100090/12/Z). The funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Ashok Venkitaraman and Paul French for critical advice and guidance on the project. We thank Chiara Cossetti and Gabriela Grondys-Kotarba in the Cambridge Institute for Medical Research Flow Cytometry facility for excellent support. We thank Liam Cassiday, Thomas Miller, and Gianmarco Contino for proofreading the manuscript.

1. Differential Fluorescent Cell Labelling
<ol>
	<li>Gene tagging with CRISPR Cas9
	<ol>
		<li>Use CRISPR Cas9 genome editing to tag rootletin (or other genes of interest) with the fluorescent proteins meGFP or mScarlet-I in human cancer cell lines. 
		NOTE: Detailed protocols for genome editing are covered elsewhere24,25,26.</li>
	</ol>
	</li>
	<li>Fluorescent dye labelling
	<ol>
		<li>Grow Cal51 human c...

<ol>
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I. Microscopic observation of giant polynuclear cell formation.</a> Experimental Cell Research. 26, 98-107 (1962).</li><li>Zimmermann, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electric+field-mediated+fusion+and+related+electrical+phenomena.">Electric field-mediated fusion and related electrical phenomena.</a> Biochimica et Biophysica Acta. 694 (3), 227-277 (1982).</li><li>Hu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusion+of+cells+by+flipped+SNAREs.">Fusion of cells by flipped SNAREs.</a> Science. 300 (5626), 1745-1749 (2003).</li><li>Bahadori, A., Lund, A. R., Semsey, S., Oddershede, L. B., Bendix, P. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+using+the+CRISPR-Cas9+system.">Genome engineering using the CRISPR-Cas9 system.</a> Nature Protocols. 8 (11), 2281-2308 (2013).</li><li>Yang, J., Shen, M. 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We demonstrate a facile and cost-effective protocol for fusing cells and visualizing the subsequent architecture of cell hybrids with microscopy, taking approximately two days from start to finish. Critical parts of this protocol are the enrichment of fused cells by cell sorting (protocol section 3), and careful validation of fused cells by microscopy (protocol section 4). These sections ensure that fused cells are readily obtained and are bona fide heterokaryons. Concentrations and incubation times should be adhered to....

Homeostatic maintenance of cellular structure is critical to life. Cells have characteristic morphologies, sub-cellular organelle numbers, and internal biochemical composition. Understanding how these fundamental properties are generated and how they go awry during disease requires laboratory tools to perturb them.
Cell fusion is the merging of two or more separate cells. Cell fusion may have been critical to the emergence of eukaryotic life1. In the human body, cell fusion is relatively rare, occurring during restricted developmental circumstances and tissue types, such as during fertilization or the formation of muscle, bone and the placenta2. This protocol describes the induction of cell-cell fusion in tissue culture cell lines with differentially fluorescently labelled organelles, as a tool to understand the mechanisms controlling cell structure and function.
In vitro induced cell-cell fusion is central to the production of monoclonal antibodies3, an important tool for biological research and disease treatment. Cell fusion has also been used to ask many different fundamental cell biological questions about cell cycle dominance4, aneuploidy5,6, cellular reprogramming7,8, the repair of damaged neurons9, viral proliferation10, apoptosis11, tumorigenesis12, cytoskeletal dynamics13, and membrane fusion14,15. Laboratory based methods to induce cell-cell fusion16,17,18,19 induce lipid membrane coalescence through the physical merging of two bilayers into one. Cell fusion can be induced by electricity18, viral based methods17, thermoplasmonic heating20, transgene expression19, and chemicals including polyethylene glycol (PEG)16,21,22.
Centrosomes are microtubule organizing centers controlling cellular shape, motility, polarization, and division23. Centrosomal roots are fibrous structures extending from centrosomes containing the protein rootletin24 (encoded by the gene CROCC). We recently used cell-cell fusion to understand how centrosome position and number varies inside heterokaryons relative to parental cells24. The rationale behind the use of this method is to track the cell of origin of roots within a heterokaryon after fusion of differentially fluorescently tagged parental cells, and thus to image organelle fusion and fission. The fluorescently tagged proteins rootletin-meGFP or rootletin-mScarlet-I are created by genome editing in separate cell lines which are then fused by PEG-mediated cell fusion. We describe the use of cell dyes (Table of Materials) to identify fused cells by flow cytometry and subsequent fluorescence microscopy identification of centrosome cell of origin and morphology (Figure 1). This approach is a robust and unique method to study how major changes in cellular state including organelle number impinge upon cell homeostasis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 ml tube</td>
			<td>Sarstedt</td>
			<td>62554502</td>
			<td></td>
		</tr>
		<tr>
			<td>37% formaldehyde solution</td>
			<td>Sigma-Aldrich</td>
			<td>F8875</td>
			<td></td>
		</tr>
		<tr>
			<td>880 Laser Scanning Confocal Airyscan Microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>8-well imaging dishes</td>
			<td>Ibidi</td>
			<td>80826</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFP alpaca GFP booster nanobody</td>
			<td>Chromotek</td>
			<td>gba-488</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Influx Cell Sorter</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Filters (70um)</td>
			<td>Biofil</td>
			<td>CSS010070</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrace Far Red</td>
			<td>ThermoFisher Scientific</td>
			<td>C34572</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrace Violet</td>
			<td>ThermoFisher Scientific</td>
			<td>C34571</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM), high glucose, GlutaMAX, pyruvate</td>
			<td>ThermoFisher Scientific</td>
			<td>31966021</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>FluoTag-X2 anti-mScarlet-I alpaca nanobody</td>
			<td>NanoTag Biotechnologies</td>
			<td>N1302-At565</td>
			<td></td>
		</tr>
		<tr>
			<td>L15 CO2 independent imaging medium</td>
			<td>Sigma-Aldrich</td>
			<td>21083027</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol red free DMEM, high glucose</td>
			<td>ThermoFisher Scientific</td>
			<td>21063029</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (1 x PBS)</td>
			<td></td>
			<td></td>
			<td>8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2HPO4, dH2O up to 1L</td>
		</tr>
		<tr>
			<td>Polyethylene Glycol Hybri-Max 1450</td>
			<td>Sigma-Aldrich</td>
			<td>P7181</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene tubes</td>
			<td>BD Falcon</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher BioReagents</td>
			<td>BP151</td>
			<td>nonionic surfactant</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>T4049</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher BioReagents</td>
			<td>BP337</td>
			<td>nonionic detergent</td>
		</tr>
	</tbody>
</table>

cell-cell fusion of genome edited cell lines for perturbation of cellular structure and function

Life is spatially partitioned within lipid membranes to allow the isolated formation of distinct molecular states inside cells and organelles. Cell fusion is the merger of two or more cells to form a single cell. Here we provide a protocol for cell fusion of two different cell types. Fused hybrid cells are enriched by flow cytometry-based sorting, followed by fluorescence microscopy of hybrid cell structure and function. Fluorescently tagged proteins generated by genome editing are imaged inside fused cells, allowing cellular structures to be identified based on fluorescence emission and referenced back to the cell type of origin. This robust and general method can be applied to different cell types or organelles of interest, to understand cellular structure and function across a range of fundamental biological questions.

Appropriately labelled cells are visible during flow cytometry by fluorescence signal higher than unlabeled control cells (Figure 2A). Gates are set for sorting of double positive cells, enriching this population directly into imaging dishes for further microscopic analyses. Fused cells are detectable as distinct double fluorescently positive cells and constitute about ~1% of the population.
<p class="jove_content" fo:keep-together.within...

Watch this Scientific Journal Video about Cell-cell Fusion of Genome Edited Cell Lines for Perturbation of Cellular Structure and Function at JoVE.com

Cell-cell Fusion of Genome Edited Cell Lines for Perturbation of Cellular Structure and Function

The purpose of this protocol is to fuse two different cell types to create hybrid cells. Fluorescence microscopy analysis of fused cells is used to track the cell of origin of cellular organelles. This assay can be used to explore how cellular structure and function respond to perturbation by cell fusion.

Life is spatially partitioned within lipid membranes to allow the isolated formation of distinct molecular states inside cells and organelles. Cell fusion ...

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